GENERATION OF ACTIVE [NIFE] HYDROGENASE IN-VITRO FROM A NICKEL-FREE PRECURSOR FORM

The maturation process of [NiFe] hydrogenases includes formation of th e nickel metallocenter, proteolytic processing of the large subunit, a nd assembly with the other hydrogenase subunit(s). An in vitro system for the maturation of the large subunit (HycE) of hydrogenase 3 of Esc herichia coil leading to an active enzyme was established. The system is based on extracts of an E. coil mutant lacking the nickel-specific transport system (nik). HycE was present in these extracts in the C-te rminally extended precursor form devoid of nickel. Addition of nickel led to nickel incorporation and proteolytic processing of HycE. Under anaerobic conditions, hydrogenase 3 activity was subsequently generate d. The maximal rate of the processing reaction was reached at a nickel concentration of 400 mu M, The accessory proteins known to be involve d in the maturation of HycE in vivo, namely HypB, HypC, HypD, HypE, Hy pF, and the protease HycI, are required for the in vitro reaction, sin ce processing of HycE did not occur in extracts of double mutants affe cted in the nik system and in one of the accessory genes. Processing o f HycE and generation of hydrogenase 3 activity were achieved in extra cts of the nik(-) Delta hycl mutant by addition of both nickel and pur ified HycI protease.